Proteins are needed for a variety of uses, especially for commercial food and chemical production and for medically therapeutic uses. These proteins may not be readily isolated from their natural sources, or may be needed in a mutated, or non-naturally occurring form. In such cases expression of heterologous proteins from cellular expression systems has proven useful.
Cellular expression systems rely on host cells, such as bacterial, yeast, fungal, and mammalian cells for expression of heterologous proteins. Bacterial cells provide an advantage in terms of ease of cultivation on inexpensive growth media and ease of manipulation of heterologous gene expression vectors. Heterologous proteins produced in bacterial expression systems can be isolated from inclusion bodies produced within the bacteria. They can also be isolated from the periplasmic space if the heterologous protein has been expressed in conjunction with a signal recognition sequence, or leader sequence, that directs the protein through the bacterial secretory pathway. Bacterial systems have been found to be disadvantageous, however, for expression of certain eukaryotic proteins, because they do not provide the intracellular machinery for appropriate post-translational modification of the proteins. Bacterial expression systems are also not the system of choice in many instances because some bacterial products, such as the bacterial lipopolysaccharide (LPS), are toxic to humans.
Mammalian expression cell systems produce appropriately modified proteins. They are not the systems of choice for producing many proteins, though, because they generally require the use of immortalized cell lines that also include proteins that transform cells, and because they are more expensive to maintain.
Yeast expression systems provide the same ease of cultivation on inexpensive growth media as do bacteria. They also provide the same ease of manipulation of heterologous expression vectors. They are advantageous over bacterial expression systems because they allow expression of proteins with appropriate post-translational modifications, such as proteolytic processing, folding, disulfide bridge formation, and glycosylation. Yeasts have also proven to be safe for human consumption through years of use in food and beverage production. Yeasts have also been used for large-scale production of human, animal, viral, and plant proteins since the early 1980s. Several species of yeast have been used to produce these foreign proteins, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, and Yarrowia lipolytica. 
Saccharomyces cerevisiae, with its long history of use in the production of bread and alcoholic beverages, is considered a safe organism for human consumption and has been more thoroughly characterized than any other species. It has therefore been the species of choice for many expression systems. A transmembrane receptor, rat somatostatin receptor type 2, as well as a surface protein, Pfs25 of Plasmodium falciparum, are examples of a large number of proteins that have been expressed in a Saccharomyces expression system.
Both intracellular and extracellular yeast expression systems have been used for heterologous protein production. Intracellular expression is preferred for proteins that are expressed in the cytoplasm, or for secreted proteins with few or no disulfide bonds. Extracellular expression, or secretion of heterologous proteins into the culture medium, provides the advantage of avoiding toxicity caused by accumulated protein and also allows for simple purification procedures. Passage of the heterologous protein through the secretory pathway also allows the heterologous protein to be post-translationally modified.
In yeast and higher eukaryotic cells Sec18p and NSF, respectively, can interact with SNARE proteins to stimulate secretory traffic in the cell. In yeast, Sec18p works in conjunction with Sec17p to promote protein secretion through sequential membrane vesicle fusion, while in mammalian cells the Sec18p counterpart, N-ethylmaleimide-sensitive factor (NSF), works in conjunction with the Sec17p mammalian counterpart, alpha-SNAP, to accomplish the same result. The secretory pathways of the two systems are remarkably similar, as indicated by the sequence identity between the proteins associated with each system and the fact that the yeast and mammalian proteins are, to a great extent, interchangeable.
Sec18p has 67% sequence identity with squid NSF, and squid NSF has approximately 75% homology with NSF found in Chinese hamster ovary cells (CHO-NSF) and Drosophila NSF (d-SNF1). Schweizer, et al., Science (1998) 279: 1203. Furthermore, when Sec18p is introduced into mammalian cells, it has been shown to interact with synaptic SNARE proteins and to synergize with alpha-SNAP to stimulate exocytosis in those cells, demonstrating that the proteins associated with membrane fusion and protein secretion are conserved from yeast to mammals. Steel, G. J., et al., Biochemistry (1999) 38(24): 7764.
The yeast Sec17 gene product has been shown to be functionally equivalent to the alpha-SNAP protein, exhibiting the exact biochemical properties expected for a yeast homolog of the mammalian transport factor. Griff, I. C., J. Biol. Chem (1992) 267: 12106. Yeast cytosol can support mammalian endosomal vesicle fusion, demonstrating that there is conservation between yeast and mammalian cells in terms of cytosolic components required for vesicle fusion. Woodman, P. G., Yeast (1996) 12: 1251.
Unfortunately, production of proteins in S. cerevisiae, either intracellularly or through the secretory pathway, results in a limited yield. In cytoplasmic expression systems, this problem has been addressed by fusing the target protein to a stable protein such as human superoxide dismutase (SOD) or human gamma interferon (IFNγ). A ubiquitin fusion expression system has also been used to increase yield and protein stability.
For secreted proteins, yield has been increased by using methylotrophic yeast, such as Pichia pastoris or Hansenula polymorpha, as host cells. Although these species have been shown to produce increased amounts of some proteins, particularly human serum albumin (which is secreted at 4 g/l by P. pastoris), protein secretion still does not regularly occur at a level that makes any of the yeast species efficient sources for heterologous protein production. Furthermore, expression in methylotrophic yeast requires the use of methanol, a highly volatile substance, in the culture environment.
In some cases, overexpression of certain proteins in conjunction with the target heterologous protein has been effective for increasing target protein secretion. Human leukocyte protease inhibitor, for example, has been shown to be secreted at a three- to four-fold higher rate when ubiquitin was simultaneously overexpressed from a chromosomal UB14 gene under the control of the GAL1 promoter. Nevertheless, those of skill in the art continue to search for host cells, expression vectors, and methods for increasing protein production and secretion in cellular expression systems.
Therefore, there is a continuing need for improved cellular expression systems that produce higher yields of heterologous proteins and can be subjected to relatively easy production and purification techniques.